P-450s are a group of hemoproteins that are the terminal components of the mixed function monooxygenase system. These enzymes are products of a gene superfamily and up to 50 distinct forms may exist in mammals. The bulk of P-450s are expressed in liver as enzymes responsible for the metabolism of foreign chemicals. In some cases endogenous steroid catabolism is associated with distinct species of P-450s; however, the physiological role of these reactions is questionable. Of principal importance is the participation of P-450s in chemical carcinogen activation. The general method for study of P-450 enzymology is through standard purification and assay of the enzymes. Two major drawbacks to this approach are the difficulty in obtaining highly purified preparations and the fact that these membrane-bound enzymes must be reconstituted with artificial lipid bilayers and the enzyme NADPH-P-450-oxidoreductase. Further human P-450s are even more difficult to purify based on the paucity of an available tissue source. We have been studying the enzymology of rat and human P- 450s by using cDNA-directed expression. Several P450s have been expressed using a variety of expression systems. Rat IIAI, IIDI and IVAI and human IIC8, IIC9, IIDI and IIAI P450s have been expressed using the COS-cell system. Mouse IAI and IA2, and rat IIAI and IVAI were expressed using the T7- vaccinia virus system. Rat IIAI and IVAI were expressed using a yeast expression system. These enzymes and others are also being expressed using baculo virus and retrovirus. Expressed P-450s have been evaluated for their catalytic activities toward a variety of drugs and carcinogens. DNA binding and mutagenesis assays are being used to evaluate the carcinogen activating properties of human P-450s.